Assay with reduced background

ABSTRACT

In as assay, an analyte is specifically associated with a reporter adenylate kinase, ADP is added and then formation of ATP is monitored. Prior to addition of ADP, adenylate kinase other than reporter adenylate kinase is removed. Assay apparatus comprises a solid phase on which is immobilised the analyte or an antibody specific for the analyte, a reporter composition comprising a thermostable adenylate kinase coupled to an antibody specific for the analyte, and ADP plus associated reagents for conversion of ADP into ATP by thermostable adenylate kinase.

This application is a 371 of PCT/GB00/00315 filed on Feb. 3, 2000 andpublished in English on Aug. 10, 2000.

FIELD OF THE INVENTION

The present invention relates to an assay with reduced background, amethod of assaying for an analyte, a method of reducing background in anassay and apparatus, in particular a test kit, for carrying out such anassay.

RELATED ART

ATP bioluminescence has rapidly become the method of choice for hygieneand cleanliness monitoring due to its combination of sensitivity andease of assay. A luciferin-luciferase bioluminescence assay can detectas little as 10⁻¹⁵ moles of ATP. Since an average microbial cellcontains approximately 10⁻¹⁸ moles of ATP, this gives a detection limitof only 10³ cells.ml⁻¹.

For most operations this detection level is sufficient, however, thereare applications where even greater sensitivity is required, even downto a single microbial cell. GB-A-2304892 describes such an assay usingthe ATP-forming enzyme adenylate kinase (AK). An average cell containsseveral hundred-fold less AK molecules than ATP molecules, however, in a10 minute incubation, a typical 400,000-fold amplification is achievedby detecting AK through the ATP it produces. This corresponds to thelevel of single cell detection, although in practice 10 cells.ml⁻¹ ismore readily achieved due to background AK and ATP contamination. Italso corresponds to a detection level of down to at least 10⁻²⁰ moles ofAK.

The commercial use of this extreme sensitivity is, therefore, underinvestigation. There are, however, some problems with more widespreaduse of this known AK-based assay. One is that while the assay detectsthe presence of micro-organisms, it does not differentiate between oneorganism and another. This has been overcome to a degree by the use ofbacteriophage to release AK from specific bacteria (Blasco R, Murphy MJ, Sanders M F and Squirrell D J (1998) Specific assays for bacteriausing phage mediated release of adenylate kinase. J. Appl. Microbiol.84: 661∝666).

Each micro-organism, however, requires a specific phage and contains anAK with different buffer requirements, plus temperature and pH optima.The second problem is more fundamental and is a problem for its use as ageneralised reporter enzyme. Whereas in hygiene and cleanlinessmonitoring the ubiquity of ATP and AK is beneficial, in an enzymereporter assay any unwanted background activity is detrimental. This isespecially so where the sample is greatly concentrated to maximisepotential detection.

A further problem is that the known assay is only effective formicroorganisms which contain AK; the known assay will not work withother biological material, such as viruses or other analytes, includingother biological such material that does not contain AK.

Transmissible Spongiform Encephalopathies (TSEs) is the term given for aspectrum of diseases associated with an unconventional transmissibleagent. The agent displays many virus-like features, such as strainvariation and mutation, but differs from conventional viruses in beingexceptionally resistant to heat, ultraviolet and ionising radiation andto chemical disinfectants. The TSEs are a heterogeneous group of fatalneurodegenerative disorders occurring in humans, mink, cats and ruminantherbivores. The endemic occurrence of the TSE “scrapie” in many sheeppopulations and more rarely human TSEs, such as Creutzfeldt-JakobDisease (CJD), has been known for some time. The occurrence of novelTSEs in wild populations of mule deer and elk in the United States andan outbreak of “Bovine Spongiform Encephalopathy” (BSE)” in cattle inthe United Kingdom and Europe has, however, emphasised the need forsensitive and reliable diagnostic tests and detection systems for thesediseases. More recently, however, it has become apparent that BSE hascrossed the species barrier to the human population giving rise to a newvariant TSE, generally known as “new variant CJD” (nvCJD) or “variantCJD” (vCJD).

The highest native concentrations of TSE infectivity are found in 263Kinfected hamster brain where titres as high as 10¹⁰ infectious units pergram of tissue are frequently reported.

Current immunoassays give positive signals for PrP^(Sc) from as littleas 1-10 g of TSE infectious brain tissue, e.g. by Western blotting orELISA. ELISA, however, is considerably more suitable than Westernblotting for the development of a fast and practical PrP(PrP^(c)+Prp^(Sc)) detection system. This level of detection isapproximately 10⁻¹⁴ moles of Prp^(Sc) but insufficient to detect thepresence of still infectious quantities of PrP^(Sc). Where PrP^(c) isalso included, however, the differential between the current andrequired level of sensitivity is significantly reduced. This bringscurrent immunoassays potentially into the appropriate range, but with aninadequate margin of safety.

There is currently great uncertainty regarding the numbers ofindividuals in the UK potentially or actually infected with new variantCreutzfeld-Jakob Disease (nvCJD). As a result there have been calls thatall surgical procedures should be carried out using disposableinstruments as a safeguard. Implementation has severe cost andprocedural implications, consequently an alternative means to validatedecontamination would be extremely beneficial, and would also be ofbenefit to other equipment such as meat processing equipment. Therefore,it remains a problem to provide an alternative assay for biologicalmaterial, especially prior protein, preferably of increased sensitivity.

WO 94/06933 discloses the use of a conjugate comprising a pyruvatekinase linked to an antibody in an assay for biological compounds and aprocess for the production of the enzyme-antibody conjugate.

Antibody-enzyme conjugates in which the enzyme may be thermostable havebeen disclosed in EP 0 304 934.

U.S. Pat. No. 4,584,272 describes an adenylate kinase that retains atleast 80% of its activity when incubated at 50° C. for 15 minutes.

The use of antibodies to detect prion proteins is known from, forexample, WO 97/10505. No assay is specified for the use of theseantibodies.

SUMMARY OF THE INVENTION

The present invention is aimed at addressing and overcoming or at leastameliorating these problems. A further object of specific embodiments ofthe present invention is to develop a rapid and sensitive method forassay of biological material, in particular for the detection of prionprotein PrP (PrP^(c) and PrP^(Sc))—as the presence of either isoform ina sample is indicative of the presence of residual PrP-expressing tissueand the potential for transmissible infectivity. A still further objectof specific embodiments of the present invention is to provide a methodfor assay of prion proteins that may be used in the screening ofcleaning protocols to determine their suitability for the removal of TSEagents from surfaces and delivery of recovered material for immunoassay.

Accordingly, a first aspect of the invention provides an assay for ananalyte, comprising specifically associating the analyte with a reporterkinase, adding ADP and testing for formation of ATP wherein, prior toaddition of ADP, kinase other than reporter kinase is substantiallyremoved.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation depicting steps 1-4 as set forth inExample 1.

FIG. 2 is a graphical representation depicting steps 5-8 as set forth inExample 1.

FIG. 3 is a graphical representation depicting steps 9 and 10 as setforth in Example 1.

FIG. 4 provides definitions for the symbols depicted in FIGS. 1-3.

DETAILED DESCRIPTION OF THE INVENTION

Thus, in use of an assay of the present invention, a reporter adenylatekinase is specifically associated with the analyte so that the amount ofreporter adenylate kinase is substantially in proportion to the amountof analyte present. In the absence of analyte there will be no reporteradenylate kinase associated and no signal generated. By substantiallyremoving adenylate kinase other than reporter adenylate kinase, thepresent invention has the advantage that the signal obtained is notcontaminated or otherwise adversely affected by any endogenous adenylatekinase that might have been present in a sample being tested. Byreference to removing adenylate kinase it is intended to refer toremoving adenylate kinase activity, such as by removing the adenylatekinase, or denaturing or otherwise inactivating it in situ. Furthermore,by addition of reporter adenylate kinase, the assay is of applicationfor detection of substantially any analyte and, unlike the prior art, isnot limited to detecting analytes that comprise their own adenylatekinase.

In an embodiment of the invention there is provided a method ofdetermining presence and/or amount of an analyte in a sample,comprising:—

-   -   exposing the sample to a reporter adenylate kinase coupled to a        binding agent specific for the analyte, so that the reporter        adenylate kinase is specifically associated with any analyte        present in the sample;    -   removing reporter adenylate kinase that is not specifically        associated with analyte;    -   exposing reporter adenylate kinase specifically associated with        the analyte to ADP; and    -   testing for formation of ATP;    -   wherein prior to addition of ADP adenylate kinase other than        reporter adenylate kinase is substantially removed.

Typically, the reporter adenylate kinase is coupled to an antibody thatbinds specifically to the analyte under investigation. The antibody maybe obtained using conventional techniques for identification endisolation of specific antibodies, and the assay of the present inventionis thus of application to substantially all analytes against which anantibody can be raised. This confers the advantage that the presentinvention is of considerably wider application compared to the knownAK/ATP-based assays, as the previous assays were restricted to targetanalytes that contained their own adenylate kinase.

The reporter adenylate kinase is suitably coupled to the specificbinding agent by conventional techniques. For example, there arenumerous ways of labelling immunoreactive biomolecules with enzymes(conjugation). Antibodies, the majority of antigens, and enzymes are allproteins and, therefore, general methods of protein covalentcross-linking can be adapted to the production of immunoassay reagents.The preparation of antibody-enzyme conjugates requires mild conditionsto ensure the retention of both the immunological properties of thantibody and the catalytic properties of the enzyme. Common methodsinclude, glutarald hyde coupling, the use of periodate oxidation ofglycoproteins to generate dialdehydes capable of forming Schiff-baselinkages with free amino groups on other protein molecules, and the useof heterobifunctional reagents, for example,succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

Endogenous adenylate kinase present in the analyte is substantiallyremoved or destroyed or otherwise inactivated before testing forformation of ATP is carried out. This removal step can conveniently beachieved by heating the endogenous adenylate kinase to a temperature atwhich it is denatured. Alternatively, other treatments might beappropriate to destroy the activity of the endogenous adenylate kinase,such as the use of ultrasound or extremes of pH or salt concentration.In an embodiment of the invention, the reporter adenylate kinase is athermostable enzyme and endogenous adenylate kinase is removed byheating. In a specific embodiment of the invention described in moredetail below, this denaturing step is carried out at about 90° C. for aperiod of about 10 minutes, though other temperatures and durations willbe appropriate so long as the endogenous adenylate kinase is renderedincapable of catalysing the formation of ATP and the reporter adenylatekinase retains its activity.

It is a further, preferred, step in the assay of the present inventionfor any ATP present prior to addition of ADP to be removed, therebyfurther decreasing the background noise in the assay. The removal ofendogenous ATP may be achieved by addition of an ATPase and incubationprior to adding ADP. More preferably, a thermolabile ATPase is used toremove ATP and then the thermolabile ATPase is itself destroyed by useof elevated temperature, to avoid the presence of the ATPase adverselyinfluencing the signal obtained using the thermostable, reporteradenylate kinase.

The precise order of carrying out the steps of the present invention isnot critical, provided that endogenous adenylate kinase is destroyedbefore addition of ADP and testing for the formation of ATP. Thus, themethod of the present invention can be carried out by treating a sampleto destroy its endogenous adenylate kinase, adding reporting adenylatekinase coupled to an antibody specific to the analyte, isolatingreporting adenylate kinase that is specifically associated with analyteand then adding ADP and testing for formation of ATP. Alternatively, theassay can be carried out by adding a reporter adenylate kinase coupledto an antibody specific for the analyte to a sample, isolating reporteradenylate kinase that is specifically associated with analyte,destroying any endogenous adenylate kinase that may be present and thenadding ADP and testing for formation of ATP. A further alternative is toadd reporter adenylate kinase coupled to an antibody specific foranalyte to the sample, treating the sample to destroy endogenousadenylate kinase, isolating reporter adenylate kinase specificallyassociated with analyte and then adding ADP and testing for formation ofATP.

In a specific embodiment of the invention described in more detailbelow, an assay is carried out by following the steps:—

-   -   1. An antibody specific to the analyte is immobilised on a solid        phase.    -   2. A sample is combined with the solid phase so that analyte        present in the sample can bind to the antibody.    -   3. The solid phase is washed, thereby washing away components of        the sample and retaining on the solid phase only any analyte        that has bound to the immobilised antibody.    -   4. A reporter composition is added to the solid phase, the        reporter composition comprising an antibody which is specific to        the analyte and which is coupled to a thermostable adenylate        kinase.    -   5. The solid phase is washed, thereby washing away unbound        components of the reporter composition and retaining reporter        composition that has specifically bound the analyte, the analyte        being itself bound to the immobilised antibody.    -   6. The solid phase is heated to denature any endogenous        adenylate kinase that may be present but so as not to denature        the thermostable adenylate kinase.    -   7. Optionally, a thermolabile ATPase is added to the solid phase        to remove any endogenous ATP.    -   8. Optionally, the solid phase is heated to destroy the        thermolabile ATPase of step 7.    -   9. ADP is added to the solid phase which is then tested for        presence and/or amount of ATP.    -   10. If ATP is detected, this indicates that adenylate kinase in        the reporter composition was bound to the solid phase, ie that        analyte was present in the sample.

The solid phase is suitably selected from conventional solid phases usedin immunoassays, and can for example be a microtitre well, a column, adip-stick or a bead, such as a latex or a magnetic bead. Examples offurther suitable solid supports are nitrocellulose, polyvinylchloride,polystyrene, diazotized paper, activated beads having a range ofappropriate linking agents and S.aureus protein A beads. Morethermostable supports are provided by plastics such as polypropylene,polycarbonate, polyphenylenine oxide polymethylpentene andfluoropolymers (e.g. PTFE, PFA, FEP and EFTE). The solid support canhave several forms dependent upon the type of support and the conditionsrequired. Commonly these will be microtitre plates, where eachindividual well serves as an independent incubation chamber. Similarly,membranes or sheets can be used providing lateral diffusion is limited.Alternatively, beads can be used, which enable the separate reactions tobe performed in different tubes under different conditions. Theseindividual matrix materials can be purchased in a variety of forms, asappropriate for the particular type of assay.

Firefly luciferin catalyses the oxidation of D(−) luciferin in thepresence of ATP-Mg²⁺ and O₂ to generate oxyluciferin and light. Thequantum yield for this reaction (0.88) is the highest known forbioluminescent reactions (Gould and Subramini, 1988). Fireflyluciferase, however, is relatively unstable and has, therefore, notproved readily adaptable as an immunoassay label (Kricka, 1993). Bycontrast, in the present invention, the luciferase enzyme can beoperated under its optimal conditions and is not exposed to harshtreatments such as antibody-coupling.

A number of extremely thermostable adenylate kinases have now beencharacterised (Ki and Takahisa, 1988; Lacher and Schäfer 1993; Rusnak etal., 1995) and are suitable for use in the present invention. One hasbeen cloned and overexpressed in E. coli (Bonisch et al., 1996) and thefull sequences of a range of others are now available as a result ofgenome sequencing programmes. A rapid and simple purification scheme isthus available to produce homogenous adenylate kinase. Initially athermal denaturation step can be employed to denature the bulk of E.coli proteins (−90-95%) while retaining the thermostable activity insolution.

This procedure has been successfully employed in embodiments of thepresent invention with several recombinant thermostable enzymes.Subsequently a generally applicable affinity purification procedure canbe utilised to yield th purified enzyme. This involves binding of theenzyme to a mimetic dye matrix and selective desorption with theadenylate kinase inhibitor P¹, P⁵-di(adenosin-5′)pentaphosphate (Rusnaket al., 1995). The use of stable enzymes overcomes problems associatedwith inactivation upon antibody-coupling, and also provide otherbenefits. Since the activity is extremely thermostable, once substratebinding and removal of unbound components has occurred, the temperaturecan be increased to e.g. 70-90° C., denaturing and inactivating anyresidual contaminating mesophilic adenylate kinase. Additionally, oncooling, a mesophilic ATPase (or apyras can be added to remove anyresidual ATP. This ensures that no ATP or AK background is now present.A further heat incubation inactivates the mesophilic ATPase and ADP isadded in order to generate ATP derived exclusively from the thermostableadenylate kinase. This ATP is then available for conventionalluciferin-luciferase bioluminescence detection. A potentiallycontaminating ATP signal is now only possible from three sources:non-specifically bound thermostable AK, ATP-contaminated ADP and AKcontaminated luciferase. The latter two can be eliminated by the use ofhigh purity reagents and careful handling. In each case, however,contamination would result in a positive signal, i.e. a PrP-free samplemight be determined to be PrP-containing but the opposite could notoccur.

A known thermostable adenylate kinases, Methanococcus jannaschii has avery high specific activity, namely 89 μmol of ATP mg⁻¹ min⁻¹. Thiscorresponds to a turnover number in excess of 2000 min⁻¹ and thepotential to produce more than 1.2×10⁵ molecules of ATP per molecule ofAK in an hour's incubation. Since 6×10⁸ molecules of ATP are detectableby ATP-bioluminescence then as few as 5×10³ molecules of PrP would bedetectable. This is 40-fold lower than the minimum number of PrP^(Sc)molecules identified as constituting a single infectious unit. Anadditional safety margin is provided by the presence of much higherquantities of PrP^(c) in relation to PrP^(Sc) indicating that thepresent invention exceeds the required sensitivity by several orders ofmagnitude.

As an alternative to use of an analyte-specific antibody to immobilizeanalyte on the solid phase, the solid phase may be provided with analyteimmobilised directly thereon without the presence of the first antibody.For example, the solid phase can itself be a substrate potentiallycontaminated by an amount, typically a trace amount, of analyte. This isthe case in respect of medical equipment potentially contaminated byvery small amounts of prion protein which are effectively immobilised onthe surface of the equipment. The assay is of use in testing for thepresence of the analyte for example following cleaning of the equipment.Analyte can also be immobilised non-specifically.

The method of the present invention may be carried out utilisingrelatively inexpensive equipment in a standard laboratory. Use of amethod of the present invention to determine when the level of prionprotein has been reduced to below detectable and, by extrapolation,infectious levels may be used to confirm the decontamination ofinstruments, equipment and other items potentially exposed to TSEinfectious agents, permitting their safe use.

In use of a specific embodiment of the invention, the first washing stepcan be repeated a number of times, in accordance with conventionalpractice in this field, the object being to remove from the solid phaseall components of the sample that have not bound specifically to theimmobilised antibody. Thus, if there is no analyte present in the samplethen the washing step will remove the whole of the sample and ultimatelythe assay will give no signal, indicating that no analyte was present.The antibody in the reporter composition binds to the same analyte asthe antibody immobilised on the solid phase. The antibody and thereporter composition can in fact have the same binding properties as theimmobilised antibody, though it is an alternative for the reporterantibody to bind to a different site on the same analyte. The reporterantibody is preferably selected so that the amount of reportercomposition that binds to the analyte is substantially proportional tothe amount of analyte present. The second washing step can, in line withthe first, be repeated a number of times in accordance with conventionalpractice, the object of the second washing step being to remove allcomponents of the reporter composition that have not specifically boundto analyte which itself has specifically bound to immobilised antibody.Thus, if no analyte is present on the solid phase the second washingstep is to remove all reporter composition, leading ultimately to nosignal being generated in the assay, indicating no analyte was presentin the sample under investigation.

This latter embodiment represents use of the principles of the inventionin a two antibody capture assay, sometimes referred to as a sandwichassay. The invention is similarly of application in antigen captureassays and antibody capture assays.

Thus in a further embodiment of the invention, an assay for analytecomprises specifically associating an analyte with a reporter adenylatekinase, wherein the analyte is bound to a solid phase. This embodimentmay be referred to as being of the antibody capture type. Binding of theanalyte to the solid phase can be achieved by non-specifically bindingthe analyte to the solid phase and then treating the solid phase toprevent further non-specific binding thereto—in this way, a number ofcomponents from a sample are bound to the solid phase, which componentsinclude the analyte of interest if present in the sample, and subsequenttreatment ensures that when an antibody is added to detect the analytethat antibody will only bind to the solid phase if analyte is present.

The use of heat to denature any endogenous kinase that may be presenthas been carried out in an embodiment above as step 6, though asmentioned this step can be carried out at an alternative juncture in theassay provided that it is carried out before addition of ADP. Further,ADP may be added before the ATPase provided the ATPase has no ADPaseactivity. The temperature and duration adopted are chosen so as to besufficient to denature th endogenous adenylate kinase whilst leavingintact the reporter adenylate kinase, this reporter adenylate kinasepreferably being a thermostable enzyme. In a specific embodimentdescribed below, heating to a temperature of about 90° C. for about 10minutes has been found effective. Sufficiently thermostable adenylatekinases may be found amongst a range of bacterial and archaeal generaand families. In the Bacteria, they may be produced, for example, bymembers of the genera Alicyclobacillus, Ammonifex, Aquifex, Bacillus,Caldariella, Calderobacterium, Caldicellulosiruptor, Caldocellum,Caloramator, Carboxydothermus, Chloroflexus, Clostridium,Coprothermobacter, Dictyloglomus, Fervidobacterium, Geotoga,Hydrogenobacter, Hydrogenothermophilus, Meiothermus, Petrotoga,Rhodothermus, Rubrobacter, Thermoactinomyces, Thermoanaerobacter,Thermoanaerobacterium, Thermoanaerobium, Thermobacterium,Thermobacteroides, Thermobifida, Thermobispora, Thermobrachium,Thermochromatium, Thermocrispum, Thermodesulfobacterium,Thermodesulforhabdus, Thermodesulfovibrio, Thermohydrogenium,Thermomicrobium, Thermomonospora, Thermonema, Thermonospora,Thermopolyspora, Thermosipho, Thermosphaera, Thermosyntropha,Thermoterrabacterium, Thermotoga and Thermus. Amongst the archaea, theymay be produced, for example, by members of the genera Acidianus,Aeropyrum, Archaeoglobus, Desulfurococcus, Desulfurolobus, Ferroglobus,Hyperthermus, Metallosphaera, Methanobacterium, Methanococcus,Methanopyrus, Methanothermus, Picrophilus, Pyrobaculum, Pyrococcus,Pyrodictium, Pyrolobus, Staphylothermus, Stetteria, Stygiolobus,Sulfolobus, Sulfophobococcus, Thermococcus, Thermofilum, Thermoplasmaand Thermoproteus.

It is preferred, though optional, also to carry out a step of removingendogenous ATP from the sample using a thermolabile ATPase andsubsequently destroying this latter enzyme, again conveniently usingheat. In a specific embodiment of the invention described below, anincubation of about 10 minutes has been effective using a thermolabileATPase and this enzyme has been then denatured by temperatures of about90° C. for 5 minutes. ATP can be released from cells or other cellularcomponents after heating. Therefore, it is preferred that the step ofremoving ATP is carried out after an initial heating of the sample, forexample after the step of using heat to destroy endogenous adenylatekinase.

It is further preferred to use ultrapure ADP, free of ATP, to avoid riskof background from contaminating ATP. As an alternative to the use of apre-purified ultrapure form of ADP, ATP-free ADP can be generated insitu by the addition of an essentially irreversible and strictlyATP-dependent mesophilic kinase plus its substrate, for example, yeasthexokinase and glucose. ATP present is converted to ADP and the kinaseis inactivated by heat prior to the incubation with thermostableadenylate kinase. Similarly, it is also preferred to use other reagentsform of contamination by kinase or ATP. Luciferin and luciferase cancontain adenylate kinase contamination and so it is preferred to usepurified forms of these, or recombinant forms of luciferase. Luciferinis preferably the d-isomer as the l-isomer can inhibition theluminescence reaction.

The invention is of particular application to detection of diseases suchas vCJD, which by December 1999 had resulted in approximately 50 deathsin the UK, with further cases reported in France and Ireland. Due to thelong and variable incubation period for this new disease however, thereis currently great uncertainty regarding the total numbers ofindividuals in the UK potentially or actually infected with vCJD.Affected individuals will frequently present with symptoms requiringneurological examination or may merely undergo common surgicalprocedures such as tonsillectomy or appendectomy along with the generalpopulation. A wide range of tissues, including tonsil and appendix, hasbeen shown to harbour vCJD infectivity in addition to brain and spinalcord. This gives rise to a significant potential for transmission ofinfection by exposure to contaminated surgical instruments, sincecomplete elimination of infectivity is not achievable using conventionalsterilisation procedures.

Although the nature of the responsible agent is not fully understood,infectivity appears to be associated very closely with the abnormalconformation (PrP^(Sc)) of a normal central nervous system protein(PrP^(c)), designated the “prion” protein. Although the prion is notuniversally accepted as being solely responsible for infectivity, thereis general agreement that it has an intimate association with it.Detection of prion protein is, therefore, considered to be an excellentmeasure of the potential presence of TSE infectivity. Prions have atendency to form insoluble aggregates and are highly hydrophobic. Thereis, therefore, considerable doubt as to whether they can be reliablydetached from surfaces and solubilised for detection by conventionalenzyme-linked immunosorbent assay. This is particularly important foritems like surgical instruments, where the presence of a very smallamount of residual material after attempted decontamination, could giverise to iatrogenic transmission of vCJD infection. In a specificembodiment, the invention describes an assay which permits in situdetection of the prion protein (Prion ELISA 1-3).

Since the presence of any residue containing either PrP^(c) or PrP^(Sc)indicates that the test item is not completely clean, the antibodyselected need not discriminate between the different conformers. Thisgreatly increases the range of antibodies available. The PrP^(Sc)conformation is, however, considerably more persistent and in general itis the form associated with infectivity which will be detected.

Thyroid stimulating hormone (TSH) is secreted by the anterior pituitaryof the brain. This hormone acts upon the thyroid, stimulating theproduction of the hormones T3 and T4. The level of TSH is controlled bya negative feed-back system that maintains a constant level of free TSH.Hyperthyroidism is a condition caused by reduced levels of circulatingTSH.

Diagnostic assays for the diagnosis of hyperthyroidism must be able todistinguish between hyperthyroidism and normal levels of circulatinghormone. The assays should be able to monitor a very low signal withoutinterference. In addition, assays for the measurement of circulating TSHshould have a broad dynamic range. A specific embodiment of theinvention, described below in more detail, provides an assay for a bloodhormone.

Assays for drugs of abuse are routinely used by clinical laboratories,drug rehabilitation clinics, health officials and clinical justicefacilities. The data obtained is often used to support medical-legalapplications involving custody of children. A decision to renew custodyof a child often rests on the results of urine drug analysesdemonstrating prolonged abstinence of drug abuse, by the parent. In manycountries random urine testing is mandatory in sensitive governmentposts, the armed forces and the transport industries. There is arequirement for more sensitive and rapid assays for drugs of abuse.

The principal agent produced by Cannabis sative isδ-9-tetrahdrocannabinol (THC). Only a small amount of THC is excreted inthe urine and the majority of assays are designed to detect the maininactive oxidation product, 11-nor-δ-tetrahydrocannabinol-9-carboxylicacid (11-COOH-THC). A specific embodiment of the invention, described inmore detail below, provides an assay for cannabis metabolite.

Urine is a complex medium, which exacerbates the problem ofdistinguishing a signal from that of background instrument noise. Thisis overcome in commercial assays by assigning a threshold concentration,above which a sample is considered positive, that exceeds the detectionlimit by several orders of magnitude. In practice this results in anumber of positive samples being assigned as negative as their signalsare below the assigned threshold. More sensitive assays make it easierto discriminate between positive and negative samples.

Many of the current commercial assays involve enzyme multipliedimmunoassay (Emit). This ELISA involves competition between drug in thetest sample and drug labelled with glucose-6-phosphate dehydrogenase(G6PDH). The G6PH drug conjugate is inactive when immobilised to asolid-phase comprising of an antibody specific for the drug of interest.On displacement the free drug-G6PH conjugate is detected by a change inthe optical density at 340 nm, as NAD+ is reduced to NADH and thesubstrate is glucose-6-phosphate is oxidised. A further specificembodiment of the invention provides an assay for cocaine metabolites inurine.

It is known that human papilloma virus (HPV) infection is a prerequisiteof the oncogenesis of many forms of cervical cancer. Currently cervicalsmears are screened for the presence of viral infection as a predictiveprecursor of oncogenesis. Another specific embodiment of the inventionis a rapid screen for the presence of viral infection of cervical cells.

Combinational libraries are powerful tools for drug discovery. Thesensitivity of the screening methodology is a major limit on the numberof combinations that can be screened for in a combinational library. Alibrary comprised of every combination of an hexa-peptide is composed of20⁶ possible combinations. More sensitive assays for the detection oftarget sequences would allow more extensive libraries to be screened. Ina yet further specific embodiment of the invention a thermostable AK isused to screen a combinational peptide library for a sequence that bindsa specific ligand of interest. This ligand may be a receptor or anenzyme.

Botulinum toxins are produced by the bacterial species Clostridiumbotulinum and are the causative agents of food-borne botulism. The mostsensitive accepted method for the detection of botulinum toxins is themouse lethality test. Few ELISA based assays using conventionalamplification methodology have the sensitivity required. A yet furtherembodiment of the invention describes an ELISA based assay for thedetection of botulinum neurotoxin in foods.

The present invention also provides, in a second aspect, apparatus fordetermining the presence and/or amount of analyte in a sample,comprising:—

-   -   a solid phase on which is immobilised the analyte or an antibody        specific for the analyte;    -   a reporter composition comprising a thermostable kinase coupled        to an antibody specific for the analyte; and    -   ADP plus, optionally, associated reagents for conversion of ADP        into ATP by thermostable kinase.

An optional additional component of the apparatus is a thermolabileATPase.

The components of the apparatus may be combined into a test kit fordetermining presence and/or amount of an analyte in a sample.

Testing for formation of ATP may be carried out using a number ofconventional means, including formation of colour. Particularlypreferred is the use of luciferin/luciferase reagents in combinationwith calibration curves to determine both presence and amount ofanalyte. The presence of magnesium ions is usually required forformation of ATP, and further details are provided in the prior artpublication GB-A-2304892, the contents of which are incorporated hereinby reference.

The present invention has been described in relation to the use ofkinases, in particular thermostable adenylate kinase. More generally,the invention also provides, in a third aspect, an assay for determiningpresence and/or amount of an analyte in a sample, comprising:—

-   -   exposing the sample to a detector composition, the detector        composition comprising an antibody specific to the analyte        coupled to a thermostable enzyme;    -   isolating (i) detector composition that has specifically bound        to analyte from (ii) detector composition that has not        specifically bound to analyte;    -   determining the presence and/or amount of detector composition        that has bound to analyte by adding a substrate for the        thermostable enzyme;    -   wherein prior to adding the substrate non-thermostable enzymes        are destroyed by application of heat.

The thermostable enzyme is suitably a kinase, and may be selected frompyruvate kinase, adenylate kinase and acetyl kinase. All of thesecatalyse formation of ATP from ADP and may be used with reagent such asluciferin/luciferase.

It is preferred that prior to addition of the substrate backgroundproduct is removed, which assists in reducing or limiting background inthe assay. Background product is suitably removed by the action ofenzyme or by thermal inactivation.

The third aspect of the invention also provides apparatus fordetermining presence and/or amount of analyte in a sample, comprising:—

-   -   a solid phase on which is immobilised the analyte or an antibody        specific for the analyte;    -   a reporter composition comprising a thermostable enzyme coupled        to an antibody specific for the analyte; and    -   substrate for the thermostable enzyme.

This aspect of the invention confers the advantage that the signalobtained from the thermostable enzyme is substantially not contaminatedby any background signals or background noise that may otherwise beobtained from the action of non-thermostable enzymes on the substrate.

Background signals and/or background noise are thus reduced and possiblyeven removed entirely. In use of a method of the third aspect of thepresent invention, an analyte is immobilised on a solid phase, a sampleis combined with the solid phase and then the solid phase is washed, thesolid phase is exposed to a detector composition including an antibodyspecific to the analyte coupled to a thermostable enzyme, the solidphase is then again washed, the solid phase is then heated to denaturenon-thermostable enzymes but so as not to denature the thermostableenzyme of the detector composition, and the amount of thermostableenzyme specifically bound to analyte which itself is specifically boundto the solid phase is determined by adding a substrate for thethermostable enzyme and determining how much product is then obtained.Immobilisation of the analyte can be through use of an analyte-specificantibody immobilised on the solid phase, or by directly binding theanalyte to the solid phase.

A further aspect of the invention provides a conjugate comprising anantibody conjugated to a thermostable enzyme for use in the assay of anypreceding aspect of the invention. In an embodiment of the invention,the enzyme an adenylate kinase. The antibody may suitably bind to ananalyte selected from a protein, a microorganism, a peptide, a toxin, ahormone and a metabolite. In a specific embodiment, the antibody bindsto a prion protein.

A stil further aspect of the invention lies in use of the apparatus ofthe invention or the conjugate of the invention in an assay for ananalyte.

The present invention is thus suitably employed to investigate theeffectiveness of a range of agents with potential for surface cleaningof contaminated surfaces to remove cellular material and PrP. Steel,glass and plastic surfaces can all be investigated to determine whetherany one is particularly recalcitrant to cleaning, and PTFE can be usedas a control surface for comparative purposes.

Thermostable adenylate kinases may be purified from a number ofthermophilic and hyperthermophilic microorganisms using a combination ofion exchange, gel filtration and affinity chromatography. The adenylatekinases may be cloned and expressed in E.coli in plasmid or phagelibraries. Direct expression can be screened for (after replica plating)by examining pooled colonies for thermostable adenylate kinase activityby incubation with ADP, followed by ATP bioluminescence assay.

A range of commercially available coupling reagents is available forantibody-adenylate kinase conjugation. Both the antibody and theadenylate kinase can be re-purified by affinity chromatography.

In certain uses of the invention, such as in the case that there is noendogenous adenylate kinase or no microbial contamination of the sampleor if the risk of such contamination is removed, it is optional todispense with the step of removing endogenous adenylate kinase. Themethod of the invention then comprises specifically associating theanalyte with a reporter adenylate kinase, adding ADP and testing forformation of ATP. Preferably, prior to addition of ADP, ATP issubstantially removed, for example by the use of an ATPase.

Specific embodiments of the invention are now described.

The assay of the present invention can involve the use of conventionalequipment and reagents required for known ATP/AK bioluminescence assays,supplemented by a thermal cycler (widely and inexpensively available forPCR), plus two specific enzymes, a thermolabile ATPase and athermostable adenylate kinase.

EXAMPLES Example 1 Assay for Prior Protein

Prion ELISA—1

(Reference is Made to the Attached Drawings)

1. Blocking

A standard item of potentially infectious equipment presents with adiverse range of biological material bound to the surface. This includesboth free and cellular ATP and mesophilic adenylate kinases (mAK). Asmall area of the surface is sectioned off to form a chamber (not shown,−1 ml volume) into which reagents can be added and removed. To preventnon-specific binding of the antibody-thermostable adenylate kinaseconjugate, the exposed surfaces, including the enclosed area of thesurgical instrument, are “blocked” by incubation in the presence ofbuffer containing, for example, the non-ionic detergent Tween 20 (1%v/v) in 10 mM PBS pH7 for 1 hour. The chamber is then washed twice with0.05% Tween 20 in 10 mM PBS pH7 prior to binding of theantibody-thermostable adenylate kinase conjugate.

2. Antibody Binding

The thermostable adenylate kinase from Bacillus stearothermophilus iscoupled to an affinity-purified polyclonal antibody via aheterobifunctional thiol-cleavable cross-linking agent,N-Succinimidyl-3-(2-Pyridyldithio) Propionate (SPDP). The antibody israised by standard procedures against a synthetic peptide correspondingto a conserved region of the prion protein, coupled tomaleimide-activated keyhole limpet haemocyanin. Active conjugate (50 μl)is added to the buffer in the chamber and incubated for 30 minutes atroom temperature.

3. Washing

The chamber is washed manually or by use of an automated washing devicewith six changes of buffer containing 0.2 M NaCl, 0.05% Tween 20 in 10mM PBS, pH7. These serve to remove unbound conjugate and any biologicalmaterial only loosely attached to the surface.

4. Linker Cleavage

Dithiothreitol is added to the last wash to a final concentration of 25mM and incubation at room temperature continued for 30 minutes. Thiscleaves the thermostable adenylate kinase moiety from the bound antibodyproviding a signal molecule in free solution proportional to theoriginal amount of prion protein present.

Prion ELISA 2

5. Recovery/Transfer

At this stage the thermostable adenylate kinase-containing solution isaspirated by pipette and transferred to the wells of a thermostableluminometer microtitre plate. Transfer of non-specific background ATPand mesophilic adenylate kinase also occurs, giving the potential forover-estimation of prion protein present on the original instrumentsurface.

6. Thermal Inactivation

The adenylate kinase used is thermostable. The temperature is,therefore, increased to 80° C. and maintained at this temperature for 10minutes in a microtitre plate thermal cycler. This thermally denaturesand inactivates any residual contaminating mesophilic adenylate kinaseleaving a preparation containing only the specific thermostableadenylate kinase proportional to the prion protein content of thesample.

7. ATP Hydrolysis

The plate is then cooled and 0.05 units.ml⁻¹ of adenosine deaminase andSolanum tuberosum apyrase added prior to incubation at 30° C. for 30minutes. This enzyme removes any residual ATP carried over from theoriginal sample.

8. Thermal Inactivation

The combination of steps 6 & 7 ensures that no ATP or AK background isnow present. A further heat incubation as in step 6 is then used toinactivate the mesophilic apyrase.

Prion ELISA —3

9. ATP Generation

Ultrapure ADP (0.1 mM) and free of ATP, is added along with magnesiumions (10 mM) in order to generate ATP derived exclusively from thethermostable adenylate kinase. Incubation is carried out at 80° C. for30 minutes. The ATP is then available for D-luciferin-luciferasebioluminescence detection.

10. ATP Bioluminescence

The ATP-containing wells are cooled to 25° C. and synthetic ultrapureD-luciferin and adenylate kinase-free luciferase added to aconcentration of 40 μM and 1 mg.l⁻¹ respectively. Individual wells areread for ATP-d pendent bioluminescence in a microtitre plate luminometerand the results recorded. The amount of light generated correlatesdirectly with the original amount of prion protein in the sample.

Example 2 An Assay for a Microorganism

A micro-organism is immobilized onto solid surface by non-specificallybinding sample components including the microorganism to the solidphase, treating the solid phase to prevent further non-specific bindingthereto and washing (we use a microtitre well in this case but otherknown solid phases are suitable, such as a latex bead or a magneticbead). An antibody specific to the micro-organism and coupled to athermostable adenylate kinase is introduced and allowed to bind, priorto further washing/recovery.

(In the known AK assay, sensitivity would have been limited by the levelof sample concentration possible before levels of background ATP andnon-specific AK obscured any signal).

The sample is now heated to about 90° C. for about 10 minutes in a cellextraction buffer (in a thermal cycler) to denature any endogenous AKpresent and release any ATP that may be trapped within themicro-organism. The sample is then cooled to 37° C. and a thermolabileATPase added. The sample is incubated for about 10 minutes to remove thebackground ATP, then the temperatures is raised to about 90° C. todenature the thermolabile ATPase.

Next, ADP is added and the temperature maintained at 90° C. so thethermostable adenylate kinase can convert ADP into ATP. This incubationgenerates ATP exclusively from the thermostable adenylate kinase. TheATP thus generated is then assayed by conventional ATP bioluminescenceand is directly proportional to th concentration of the target present.

Example 3 An Assay for a Microorganism

A micro-organism is captured by a conventional capture technique, usinga specific antibody immobilised onto a solid surface (we use amicrotitre well in this case but other known solid phases are suitable,such as a latex bead or a magnetic bead). After washing/recovery, asecond antibody specific to the micro-organism and coupled to athermostable adenylate kinase is introduced and allowed to bind, priorto further washing/recovery.

Thus, the method of Example 1 is repeated but using a microorganismimmobilized using antibody.

Example 4 A Blood-Hormone Assay

An antibody specific for the alpha subunit of TSH is immobilised onto asolid-phase. The solid-phase is treated to prevent further non-specificbinding thereto. The solid-phase is washed with wash buffer, optionallycontaining detergent. A test sample of blood serum is added.

The sample is then incubated, e.g.: 37° C. for 60 mins, allowing thefree TSH in the sample to bind to the capture antibody. The solid-phaseis then washed to remove non-specifically bound material and an antibodyspecific for the beta subunit of TSH is added, to which a thermostableadenylate kinase reporter enzyme has been conjugated. The conjugate isthen incubated at 37° C. for 60 minutes, or equivalent.

Non-bound material is then removed by washing and any endogenous ATPpresent on the solid-phase is removed by the addition ofadenosine-5′-triphosphatase (an alternative is apyrase). The sample isthen heated to 90 C, or equivalent, to denature and inactivate anymesophilic adenylate kinase that may be present.

Adenosine diphosphate (ADP) is added and the temperature is maintainedat 90 C so that the thermostable adenylate kinase can convert the ADP toATP. This incubation generates ATP exclusively from thermostableadenylate kinase. The ATP generated is then assayed by conventional ATPbioluminescence technology using a luciferin/luciferase reaction. Signalfrom contaminating adenylate kinase in the luciferin/luciferase reagentsmay be quenched by the addition of a specific enzyme inhibitor. The ATPbioluminescence measured is directly proportional to the concentrationof the TSH in the original test sample.

Whilst the solid-phase used in the above is a microtitre-plate, othersolid-phases are suitable, such as latex or magnetic bead. The testsample may be whole blood or other body fluid, rather than blood, andthe antibody may be a polyclonal or a monoclonal antibody.

Example 5 An Assay for Cocaine Metabolites in Urine

A thermostable G6PDH is used as reporter enzyme. Test antibody specificfor the class of drug of interest is immobilised onto a micro-titreplate as solid-phase. The solid-phase is treated to prevent furthernon-specific binding thereto. The solid-phase is washed with washbuffer, which may or may not contain detergent. A test sample of urineis added along with the drug-G6PDH conjugate. The drug-G6PDH isthermostable and is not active when bound to the antibody immobilised tothe solid-phase.

The sample is then incubated, at 37° C. for 60 mins. The contents of themicro-titre well is then removed and heated to 90° C. to inactivate anymesophilic G6PDH present. The temperature is then maintained at 90° C.and the substrate glucose-6-phosphate and cofactor NAD+ is added in theappropriate buffer. The rate of change in the absorbance at 340 nm ismeasured and is directly proportional to the level of drug metabolite inthe test sample.

Another reporter for this assay is a thermostable adenylate kinase. Testantibody specific for the class of drug of interest is immobilised ontoa solid-phase. The solid-phase is treated to prevent furthernon-specific binding thereto. The solid-phase is washed with washbuffer, which may or may not contain detergent. A urine test sample isadded along with the drug-adenylate kinase (AK) conjugate. The drug-AKconjugate is thermostable and is not active when bound to the antibodyimmobilised to the solid-phase.

The sample is then incubated, e.g.: 37° C. for 60 mins. The contents ofthe micro-titre well is then removed and endogenous ATP removed byaddition of adenosine-5′-triphosphatase or apyrase and incubation at 37°C. The sample is then heated to 90° C. to inactivate any mesophilicadenylate kinase present.

Adenosine diphosphate (ADP) is added and the temperature is maintainedat 90° C. such that the thermostable adenylate kinase can convert theADP to ATP. This incubation generates ATP exclusively from thermostableadenylate kinase. The ATP generated is then assayed by conventional ATPbioluminescence using a luciferin/luciferase system. Signal fromcontaminating adenylate kinase in the luciferin/luciferase may bequenched by the addition of a specific enzyme inhibitor. The ATPbioluminescence measured is directly proportional to the concentrationof the drug metabolite in the original test sample.

Other solid-phases are suitable, such as latex or magnetic bead, and thetest sample may be sera or other body fluid.

Example 6 Assays for the Detection of Human Papilloma Virus DNA

Assay A: Cervical cells are collected and resuspended in phosphatebuffered saline. PCR amplification of the HPV16, or equivalent sequence,is carried out as described in Lambropoulous et al. (1994) Journal ofMedical Virology: 43, 228-230 using the consensus primers MY11 and MY09and 30 rounds of amplification.

The PCR products are then transferred and immobilised on to anon-charged nylon coated microtitre plate, or equivalent. Anoligonucleotide probe specific for HPV16 (MY14) conjugated to athermostable adenylate kinase is then added and incubated. Theoligonucleotide-AK conjugate is prepared following an identical methoddescribed the synthesis of DNA-antibody conjugates. This complexcomprises of a biotinylated AK and an avidin-biotinylated DNA complexgenerated using available methodology: Ruzicka et al. Science 1993, 260,698-699.

Non-bound material is then removed by washing and any endogenous ATPpresent on the solid-phase is removed by the addition ofadenosine-5′triphosphatase or apyrase. The solid-phase is then washedand the sample heated to 90° C., or equivalent, to denature andinactivate any mesophilic adenylate kinase that may be present.

Adenosine diphosphate (ADP) is added and the temperature is maintainedat 90° C. such that the thermostable adenylate kinase can convert theADP to ATP. This incubation generates ATP exclusively from thermostableadenylate kinase. The ATP generated is then assayed by conventional ATPbioluminescence using a luciferin/luciferase reaction. A positive signalis indicative of HPV infection.

Assay B: Cervical cells are collected and fixed onto a solid-surface, anon-charged nylon membrane contained within a microtitre plate. Thecells are lysed and the endogenous ATP present on the solid-phase isremoved by the addition of adenosine-5′-triphosphatase or apyrase. Anoligonucleotide probe specific for HPV16 (MY14:5′CATACACCTCCAGCACCTAA3′) conjugated to a thermostable adenylate kinaseis then added. The oligonucleotide-AK conjugate is prepared following anidentical method described the synthesis of DNA-antibody conjugates.This complex comprises a biotinylated AK and an avidin-biotinylated DNAcomplex generated using available methodology: Ruzicka et. al. Science1993, 260, 698-699.

After incubation, 37° C. for 60 min, the sample is heated to 90° C., orequivalent, to denature and inactivate any mesophilic adenylate kinasethat may be present. ADP added and the temperature is maintained at 90°C. such that the thermostable adenylate kinase can convert the ADP toATP. This incubation generates ATP exclusively from thermostableadenylate kinase. The ATP generated is then assayed by conventional ATPbioluminescence using a luciferin/luciferase reaction. A positive signalis indicative of HPV infection.

Example 7 An Assay to Screen Peptide Combinational Libraries

Peptides are synthesised on small beads (100 μl-200 μm) using standardsolid-phase peptide synthesis methodology. The sequence corresponds to acombinational peptide library generated as described Lam. et al. (1991)Nature (UK). 354, 82-84.

The beads are split into 20 portions and a separate amino acid coupledto each portion. The beads are then recombined, randomised, and splitinto 20 for addition of the next amino acid. This process is repeated tobuild a peptide library of all possible combinations of amino acids. Intheory each bead should have a different peptide sequence attached.After synthesis the beads are washed and any endogenous ATP is removedby addition of adenosine-5′-triphosphatase or apyrase. Aligand-thermostable AK conjugate is added and the sample heated to 90°,or equivalent, to denature and inactivate any mesophilic adenylatekinase that may be present.

Adenosine diphosphate (ADP) is added and the temperature is maintainedat 90° C. such that the thermostable adenylate kinase can convert theADP to ATP. The beads are split into portions and screened for thegeneration of light generated by a luciferin/luciferase reaction using astandard luminescence reader. Portions generating a positive signal aresplit into further portions and re-screened. This process is continuedusing a microscope equipped with a charge couple device camera, untilthe signal from a single bead is identified. The bead is removed and thesequence of peptide is then determined using standard micro-sequencingmethodology.

Example 8 An Assay for Botulinum Toxin

Antibody specific for the botulinum toxin is immobilised onto asolid-phase. The solid-phase may be a microtitre-plate but othersolid-phases are suitable, such as latex or magnetic bead. Thesolid-phase is treated to prevent further non-specific binding thereto.The solid-phase is washed with wash buffer, which may or may not containdetergent. Test sample is added. The test sample is a food sample, butmay be whole blood or body fluid. The sample is then incubated, e.g.:37° C. for 60 mins, allowing the free toxin in the sample to bindcapture antibody. The solid-phase is then washed to removenon-specifically bound material and an antibody specific for thebotulinum toxin is added to which a thermostable adenylate kinasereporter enzyme has been conjugated. This antibody may be a polyclonalor a monoclonal antibody. The conjugate is then incubated at 37° C. for60 minutes, or equivalent.

Non-bound material is then removed by washing and any endogenous ATPpresent on the solid-phase is removed by the addition ofadenosine-5′-triphosphatase or apyrase. The solid-phase is washed andthe sample heated to 90° C., or equivalent, to denature and inactivateany mesophilic adenylate kinase that may be present.

Adenosine diphosphate (ADP) is added and the temperature is maintainedat 90° C. such that the thermostable adenylate kinase can convert theADP to ATP. This incubation generates ATP exclusively from thermostableadenylate kinase. The ATP generated is then assayed by conventional ATPbioluminescence using a luciferin/luciferase reaction. Signal formcontaminating adenylate kinase in the luciferin/luciferase may bequenched by the addition of a specific enzyme inhibitor. The ATPbioluminescence measured is directly proportional to the concentrationof the toxin in the original test sample.

The invention thus provides method and apparatus for a sensitivecapture-type assay.

REFERENCES

-   Gould S J and Subramini S (1988) Firefly luciferase as a tool in    molecular and cell biology. Anal. Biochem. 175: 5-13.-   Kricka L J (1993) Ultrasensitive immunoassay techniques. Clin.    Biochem. 26: 325-331.-   Ki W-K and Takahisa O (1988) Purification and characterisation of    adenylate kinase from extreme thermophile Thermus caldophilius GK24.    Korean J. Appl. Microbiol. Bioeng. 16: 393-397.-   Lacher K and Schäfer G. (1993) Archaebacterial adenylate kinase from    the thermoacidophile Sulfolobus acidocaldarius: purification,    characterization and partial sequence. Arch. Biochem. Biophys. 302:    391-397.-   Rusnak P, Haney P and Konisky J (1995) The adenylate kinases from a    mesophilic and three thermophilic methanogenic members of the    archaea. J. Bacteriol. 177: 2977-2981.-   Bonisch H, Backmann J, Kath T, Naumann D and Schäfer G (1996)    Adenylate linase from Sulfolobus acidocaldarius: expression in    Escherichia coli and characterization by Fourier transfrom infrared    spectroscopy. Arch. Biochem. Biophys. 333: 75-84.

1. An assay for an analyte, comprising contacting the analyte with athermostable reporter adenylate kinase coupled to a binding agentspecific for the analyte, wherein a complex is formed, adding ADP andtesting for formation of ATP wherein, prior to the addition of ATP,endogenous kinase and uncomplexed thermostable reporter adenylate kinaseis substantially removed by washing and, residual endogenous kinase isinactivated by heating, wherein the amount of said ATP formed correlatesto the concentration of the analyte.
 2. The assay of claim 1, whereinthe amount of thermostable reporter adenylate kinase complexed with theanalyte is substantially proportional to the amount of analyte.
 3. Theassay of claim 1, wherein formation of ATP is measured usingluciferin/luciferase.
 4. An assay for determining the presence and/oramount of an analyte in a sample, comprising exposing the sample to athermostable reporter adenylate kinase coupled to a binding agentspecific for the analyte, so that the reporter adenylate kinase isspecifically associated with any analyte present in the sample via thebinding agent; removing thermostable reporter adenylate that is notbound to analyte; exposing said thermostable reporter adenylate kinasebound to the analyte to ADP; and testing for formation of ATP, whereinprior to addition of ADP, residual kinase other than thermostablereporter adenylate kinase is substantially removed by heating.
 5. Theassay of claim 1, comprising further adding an ATPase to the analyte andremoving the ATPase from the analyte prior to adding ADP.
 6. The assayof claim 5, wherein the ATPase is inactivated by heating the ATPase. 7.An assay for determining presence and/or amount of an analyte in asample comprising: exposing the sample to a detector compound, thedetector compound comprising an antibody specific to the analyte coupledto a thermostable enzyme; isolating (i) detector compound that hasspecifically bound to analyte from (ii) detector compound that has notspecifically bound to analyte; determining the presence and/or amount ofdetector compound that has bound to analyte by adding a substrate forthe thermostable enzyme and measuring a product formed by conversion ofsaid substrate to said product by said thermostable enzyme; thereinprior to adding the substrate non-thermostable enzymes are destroyed byapplication of heat.
 8. The assay of claim 7, wherein the enzyme isadenylate kinase and the substrate is ADP, the ADP is converted into ATPby the thermostable enzyme.
 9. The assay of claim 8, wherein backgroundATP compound is removed by the addition of ATPase prior to adding ADP.10. An assay for an analyte, comprising the steps: (a) specificallybinding the analyte with a thermostable reporter kinase which has beencoupled to a binding agent specific for the analyte forming a complex;(b) washing to remove endogenous-non-thermostable kinase andthermostable reporter kinase not bound to analyte; (c) heating toinactivate endogenous kinase not removed by step (b); and (d) adding ADPand testing for formation of ATP.